Combination of benzoquinazoline antifolates and protecting agents

ABSTRACT

The use of a protecting agent, for example a folate derivative such as folic acid or leucovorin, in combination with a non-competitive folic acid analogue, for example benzoquinazoline derivatives, for use in reducing the side effects associated with the administration of such non-competitive folic acid analogues, and pharmaceutical formulations comprising such combinations are disclosed.

This application is a continuation of U.S. Ser. No. 08/448,393 filed onJun. 6, 1995 now U.S. Pat. 6,358,952 B1, which is a 371 ofPCT/G893/02611, filed Dec. 21, 1993.

The present invention relates to novel combinations of non-competitivefolic acid analogues with protecting agents, and to methods of treatmentusing these combinations.

European Patent Application 0505640 provides a method for improving thetherapeutic utility of GAR-transformylase inhibitors and otherantifolates by co-administering a folate binding protein binding agentto the host undergoing treatment.

Thymidylate synthase is an enzyme catalysing the terminal step in the denovo synthesis of thymidylate required for DNA synthesis. It has beenpostulated that inhibitors of this enzyme may be expected to haveanti-tumour activity and it has been reported (Jones et al, J. Med.Chem. 1986, 29, 468) that the in-vivo antitumour activity ofN¹⁰-propargyl-5,8-dideazafolic acid arises solely from its inhibitoryeffect on this enzyme.

PCT publication WO/91/19700 discloses the compound of the formula (I)

or a salt thereof, wherein the dotted line represents a single or doublebond.

R¹ is C₁₋₄ alkyl or amino optionally substituted by a C₁₋₄ alkyl C₁₋₅alkanoyl or benzyl group;

R², R³, R⁴ and R⁵ are the same or different and each is selected fromhydrogen, phenyl, halo, nitro.

a group S(O)_(n) R⁸ wherein n is the integer 0, 1 or 2 and R⁸ is halo oris C₁₋₄ alkyl or a group NR⁹R¹⁰ wherein R⁹ and R¹⁰ are both hydrogen,

a group NR¹¹R¹² wherein R¹¹ and R¹² are the same or different and eachis hydrogen or C₁₋₄ alkyl.

a group OR¹³ wherein R¹³ is hydrogen or C₁₋₄ alkyl optionallysubstituted by halo;

a C₁₋₄ aliphatic group optionally substituted by a group OR¹⁴ or NR¹⁴R¹⁵wherein R¹⁴ and R¹⁵ are the same or different and each is hydrogen orC₁₋₄ alkyl:

or two of R² to R⁵ are linked together to form a benzo group.

or one of R² to R⁵ is a group —X—Y—R¹⁶ wherein X is CH₂, NR¹⁷, CO orS(O)_(m) and m is 0, 1 or 2 and R¹⁷ is hydrogen or a C₁₋₄ aliphaticgroup and Y is CH₂, O, NR¹⁷′ or S(O)_(m′) wherein m′ is 0, 1 or 2 andR¹⁷ is hydrogen or a C₁₋₄ aliphatic group provided that X and Y are onlythe same when each is CH₂, or —X—Y— is a group —O—. —NR¹⁷—, —CH═CH— or—N═N— wherein R¹⁷ is as hereinbefore defined, R¹⁶ is a C₁₋₄ aliphaticgroup or a 5- or 6-membered aromatic ring optionally substituted by agroup R¹⁸ at a position at least one carbon atom removed from thatlinked to Y, the 5- or 6-membered ring being optionally furthersubstituted by a halo atom; and R¹⁸ is halo, C₁₋₄ alkoxy, nitro,nitrile, C₁₋₄ alkyl optionally substituted by halo, halo or a groupCOR¹⁹ wherein R¹⁹ is hydroxy, C₁₋₄ alkoxy or C₁₋₆ alkyl optionallysubstituted by one or two carboxyl groups or C₁₋₁₂ esters thereof or R¹⁹is a group NR²⁰R²¹ wherein R²⁰ and R²¹ are the same or different andeach is hydrogen or C₁₋₄ alkyl optionally substituted by hydroxy or R¹⁹is an amino acid group or an ester thereof in which the first nitrogenatom of the amino acid group may be linked to the 5- or 6-memberedaromatic ring to form a further 5- or 6-membered heterocyclic ring orR¹⁹ is an C₂₋₃ alkylene croup linked to the 5- or 6-membered aromaticring to form a further 5- or 6-membered ring;

R⁶ and R⁷ are the same or different and each is hydrogen, C₁₋₄ alkyloptionally substituted by hydroxy or C₁₋₄ alkoxy or together form abenzo group;

provided that at least one of R² to R⁷ is other than hydrogen and thatR⁴ is not methoxy when R¹ is hydroxy or methyl is an inhibitor of theenzyme thymidylate synthase and has anti-tumour activity.

By the term halo is meant fluoro, bromo, chloro and iodo.

By the term C₁₋₄ aliphatic group is meant a C₁₋₄ alkyl, alkenyl, oralkynyl group.

By the term amino acid group is meant naturally occurring amino acids.

Preferred amino acid groups include glycine, glutamic acid andpolyglutamic acid groups.

When the amino acid group is linked to the 5- or 6-membered aromaticring, this is via a carbon atom of the aromatic ring adjacent to carbonto which COR¹⁹ is attached.

Preferably the dotted line is a double bond.

Suitable substituents for the aromatic ring R¹⁶ include halo, C₁₋₄ alkyland C₁₋₄ alkoxy each optionally substituted by one to five halo atoms.Most suitably there are one or two substituents selected from fluoro,chloro, methyl, trifluoromethyl and methoxy, and preferably fluoro, orno substituents on the aromatic ring. In one preferred embodiment,—X—Y—R¹⁶ is a group

wherein R¹⁸ is as hereinbefore defined and preferably a group COR¹⁹ ashereinbefore defined and R²² is hydrogen or fluoro.

In a further preferred embodiment X—Y—R¹⁶ is a group

wherein H₂NR^(19a) is a glutamic or polyglutamic acid group and Z isCH₂, S or O.

Suitably, R¹ is an amino group optionally substituted by one or twomethyl or ethyl groups or R¹ is a methyl or ethyl group. Preferably R¹is an amino or methyl group.

Suitably, at most only three, and preferably at most only two, of R² toR⁵ are other than hydrogen and each is independently selected fromhydrogen, halo, hydroxy, nitro, C₁₋₃ alkyl optionally substituted byhydroxy or C₁₋₂ alkoxy, C₁₋₃ alkoxy, amino optionally substituted by oneor two methyl or ethyl groups, or a group S(O)_(n) R²³ wherein n is 0, 1or 2 and R²³ is a C₁₋₄ alkyl group or an amino group optionallysubstituted by one or two methyl or ethyl groups, or one of R² to R⁵ isa group —X—Y—R²⁴ where R²⁴ is a group

wherein R¹⁸, R^(19a), R²² and Z are as hereinbefore defined. In onepreferred embodiment R¹⁸ is nitro or a group

wherein R²⁵, R²⁶ and R²⁷ are the same or different and each is hydrogenor a C₁₋₄ alkyl group and t is an integer from 0 to 6. Preferably R²⁵,R²⁶ and R²⁷ are hydrogen and t is 0. Preferably Z is CH₂ or S.

Preferably one of R² to R⁵ is a group —X—Y—R²⁴ as hereinbefore defined.Preferably R³ is a group —X—Y—R²⁴.

Suitably R⁶ and R⁷ are the same or different and each is hydrogen,methyl, ethyl or methyl substituted by bromo, hydroxy or methoxy.Preferably R⁷ is hydrogen and R⁶ is methyl.

Preferably —X—Y— is a group —SO₂NR¹⁷— or CH₂NR¹⁷ wherein R¹⁷ is ashereinbefore defined.

Suitably R¹⁷ is hydrogen or a C₁₋₄ alkyl or alkenyl group and preferablyR¹⁷ is hydrogen or methyl.

A further group of compounds of the formula I is that of the formula(II)

or a salt thereof, wherein R¹, R⁶, R⁷ and the dotted line are ashereinbefore defined and R²⁸ to R³¹ are the same or different and eachis selected from hydrogen, halo, nitro, a group S(O)_(n)R⁸, a groupNR¹¹R¹², a group OR¹³, or a C₁₋₄ aliphatic group optionally substitutedby a group OR¹⁴ or NR¹⁴R¹⁵ wherein R⁸, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵hereinbefore defined, provided that R²⁸ to R³¹ are not all hydrogen andthat R³⁰ is not methoxy wherein R¹ is hydroxy or methyl.

A preferred group of compounds of the formula (I) is that of the formula(III):

or a salt thereof, wherein R¹, R⁶ and R⁷ are as hereinbefore defined andR³² to R³⁵ are the same or different and one is a group X—Y—R¹⁶ and theothers are the same or different and each is selected from hydrogen,halo, nitro, a group S(O)_(n)R⁸, a group NR¹¹R¹² a group OR¹³ or a C₁₋₄aliphatic group optionally substituted by a group OR¹⁴ or NR¹⁴R¹⁵,wherein X, Y, R⁸, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are as hereinbeforedefined.

A further preferred group of compounds of the formula (I) is that of theformula (IV):

wherein R¹, R⁶, R⁷ and R³² to R³² are as hereinbefore defined.

Preferably R³³ is a group X—Y—R¹⁶ as hereinbefore defined.

Preferred compounds of the formula (I) include:

3-Amino-9-bromobenzo[f]quinazolin-1(2H)-one

3-Amino-9-ethynylbenzo[f]quinazolin-1(2H)-one

N-(4-((3-Amino-1,2,5,6-tetrahydro-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid

N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

N-(4-((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid

N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid

N-(4(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glumaticacid

(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino-1-oxo-2-isoindolinyl)glutaricacid

9-((4-Acetylanilino)methyl)-3-methylbenzo[f]quinazolin-1(2H)-one

3-Methyl-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one

N-(4-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glutamicacid

3-Amino-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one

9-((4-Acetylanilino)methyl)-3-aminobenzo[f]quinazolin-1(2H)-one

(RS)-2-(2-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-2-oxoethyl)glutaricacid

Ethyl4-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyrate

4-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyricacid

N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycine

EthylN-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycinate

Certain compounds of the formula (I) contain asymmetric carbon atoms andare, therefore, capable of existing as optical isomers. The individualisomers and mixtures of these are included within the scope of thepresent invention.

It has now been found that the compounds of formula (I) can be used incombination with a protecting agent described hereinafter to prevent ordecrease unwanted side effects of these compounds.

The present invention relates to the administration of the compounds offormula (I) in combination with a protecting agent which blocks theintestinal toxicity of the compounds of formula (I) without blockingtheir activity. Suitable protecting agents are folates, for example,folic acid, tetrahydrofolate, 5-formyltetrahydrofolate (leucovorin) or5-methyltetrahydrofolate. Other protecting agents include thymine orthymidine. The protecting agent may be administered as an oralformulation. It is within the scope of the invention to administer theprotecting agent before, during or after the administration of acompound of formula (I). Preferably, the protecting agent will be givenslightly before administration of a compound of formula (I), i.e.between 15 minutes and one hour before the compound of formula (I).

The present invention also relates to combination therapy comprising theadministration of non-competitive folic acid analogues in combinationwith protecting agents as defined above. This allows the use of thenon-competitive folic acid analogues at higher doeses than could be usedin the absence of the protecting agent as defined above, and thereforehigher, more potent doses can be achieved while minimizing hosttoxicity. The presence of the protecting agent reduces the toxicity ofthe non-competitive folic acid analogues at the doses utilized in theabsence of the protecting agent (i.e. less toxicity at the same dose inthe presence of a protecting agent).

Non-competitive folic acid analogues are defined as structural analoguesof folic acid (including but not limited to the benzoquinazolinecompounds of formula I, e.g., compounds of example 1 or 2) expressingclassical non-competitive inhibition kinetics (vs. the folate substrate)on the target enzyme (e.g., thymidylate synthase, glycineamideribonucleotide transformylase or dihydrofolate reductase).Alternatively, the non-competitive inhibitors of the present inventionare defined as structural analogues of folic acid (as defined above)which retain their tumour cell kill in the presence of the folateprotecting agents defined above, on the target enzyme. Thenon-competitive inhibitor may been given as an intravenous orintraperitoneal bolus or infusion and the protecting agent may be givenas an intravenous or intraperitoneal bolus infusion or as an oralformulation. For a definition of classical non-competitive inhibitionsee I. H. Segal (1975) Enzyme kinetics, John Wiley and Sons, which isincorporated herein by reference.

More specifically, protecting agents selected from folic acid,tetrahydrofolate. 5-formyltetrahydrofolate (leucovorin) or5-methyltetrahydrofolate can block the intestinal toxicity of compoundsof formula (I) without blocking their anti-tumor activity.

Preferably the non-competitive, folic acid analogue (i.e., non-competiveinhibitor) is a compound of formula (I) and the protecting agent isfolic acid or 5-formyltetrahydrofolate. More preferably, thenon-competitive, folic acid analogue (non-competitive inhibitor) is acompounds selected from(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid, orN-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid and the protecting agent which is used in combination is folic acidor 5-formyltetrahydrofolate. Most preferably, the compound(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxoxbenxo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid is utilized in combination with folic acid or5-formyltetrahydrofolate.

The salts of the compounds of the present invention can also be used incombination with the protecting agent or salts thereof.

Suitable salts of the compounds of the present invention or of theprotecting agents may comprise acid addition salts derived from an aminogroup or anionic species derived from a compound of formula (I) orprotecting agent, for example when this is substituted by a carboxygroup, and a cation. In both types of salts, the therapeutic activityresides in the moiety derived from the compounds defined herein and theidentity of the other component is of less importance although fortherapeutic and prophylactic purposes it is, preferably,pharmaceutically acceptable to the patient. Examples of pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitricand sulphuric acids, and organic acids, such as tartaric, acetic,trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic,gluconic, succinic and methanesulphonic and arylsulphonic, for examplep-toluenesulphonic, acids. Examples of salts comprising an anionicspecies derived from a compound of formula (I) and a cation includeammonium salts, alkali metal salts, such as sodium and potassium salts,alkaline earth salts, such as magnesium and calcium salts and saltsformed with organic bases, for example, amino salts derived from mono-,di- or tri-(lower alkyl) or (lower alkanol)amines, such astriethanolamine and diethylamino-ethylamine, and salts with heterocyclicamines such as piperidine, pyridine, piperazine and morpholine. Thepharmaceutically acceptable salts together with the salts which are notthus acceptable have utility in the isolation and/or the purification ofthe compounds of the invention, and the pharmaceutically unacceptablesalts are also useful in being convertible to the pharmaceuticallyacceptable salts by techniques well known in the art.

The route by which the compound or salt of the present invention isadministered to the animal may be oral, topical, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous or rectal). If thecompound or salt is presented in the form of a pharmaceuticalformulation, which is preferred, then the actual formulation employedwill of course depend on the route of administration elected by thephysician or veterinarian. For example, if oral administration ispreferred, then the pharmaceutical formulation employed is, preferably,one which is suitable for such a route.

A therapeutically effective amount of a compound or salt of the presentinvention will depend upon a number of factors including, for example,the age and weight of the animal, the precise condition requiringtreatment and its severity, the nature of the formulation, and the routeof administration, and will ultimately be at the discretion of theattendant physician or veterinarian. However, an effective amount of anon-competitive folic acid analogue of the present invention (asdescribed hereinbefore) to be used in combination with a protectingagent (as described hereinbefore) for the treatment of neoplasticgrowth, for example colon or breast carcinoma will generally be in therange of 0.1 to 100 mg/kg body weight of recipient (mammal) per day andmore usually in the range of 1 to 50 mg/kg body weight per day. Thus,for a 70 kg adult mammal, the actual amount per day would usually befrom 70 to 3500 mg and this amount may be given in a single dose per dayor more usually in a number (such as two, three, four, five or six) ofsub-doses per day such that the total daily dose is the same. Aneffective amount of a salt of the present invention may be determined asa proportion of the effective amount of the compound per se.

An effective amount of a compound of the present invention for thetreatment of disorders of the immune system (e.g. thermatoid arthritisand tissue rejection) and related diseases such as psoriasis, willgenerally be in the range of 0.5-10 mg/kg body weight of recipient(mammal) per day. Thus for a 70 kg adult human, the actual amount perday would usually be from about 5-700 mg per day and this amount may begiven in a single dose per day or more usually dosing would beintermittent e.g. 12 hourly intervals or weekly. An effective amount ofa salt of the present invention may be determined as a proportion of theeffective amount of the compound per se.

An effective amount of a compound of the present invention for thetreatment of bacterial and fungal infections is in the range of 0.1-100mg/kg bodyweight of recipient (mammal) per day and preferably in therange of 1-50 m/kg body weight per day. Thus, for a 70 kg adult mammal,the actual amount per day is from 70-3500 mg and this amount may begiven in a single dose per day or more preferably in a number (such astwo, three, four, five or six) of sub-doses per day such that the totaldaily dose is the same. An effective amount of a salt of the presentinvention may be determined as a proportion of the effective amount ofthe compound per se.

An effective amount of a protecting agent (e.g., folic acid) of thepresent invention for use in combination with a non-competitive, folicacid analogue (including but not limited to the compounds of the presentinvention as described herein) is in the range of 1-300 mg/kg bodyweightof recipient (mammal) per day and preferably in the range of 5-50 mg/kgbody weight per day. Thus, for a 70 kg adult mammal, the actual amountper day is from 350-3500 mg and this amount may be given in a singledose per day or more preferably in a number (such as two, three, four,five or six) of sub-doses per day such that the total daily dose is thesame.

The treatment of neoplastic growth with a compound of the presentinvention may at times require the administration to the animal of anantidote or rescue agent, e.g. thymidine.

The compounds, per se, of the present invention which can be used incombination with a protecting agent as described herein include, but arenot limited to, those disclosed in PCT publication WO/91/19700. Theprotecting agents of the present invention are readily available andwell known to those skilled in the art.

Whilst it is possible for the compounds or salts of the presentinvention to be administered as the raw chemical, it is preferred topresent them in the form of a pharmaceutical formulation.

Accordingly, the present invention further provides a pharmaceuticalformulation, for medicinal application, which comprises a compound ofthe present invention and a protecting agent or pharmaceuticallyacceptable salts thereof, as hereinbefore defined, in combination withone or more pharmaceutically acceptable carriers therefor, andoptionally one or other therapeutic agredients.

The pharmaceutical formulation may optionally contain other therapeuticagents that may usefully be employed in conjunction with the compound orsalt of the present invention, for example a pyrimidine nucleosidetransport inhibitor that is capable of enhancing the antineoplasticactivity of the compounds and salts of the presents invention. Theexpression “pharmaceutically acceptable” as used herein in relation tothe carrier is used in the sense of being compatible with the compoundor salt of the invention employed in the formulation and with any othertherapeutic agent that may be present, and not being detrimental to therecipient thereof. The carrier itself may constitute one or moreexcipients conventionally used in the art of pharmacy that enable thecompound or salt of the present invention and any other therapeuticagent that may be present, to be formulated as a pharmaceuticalformulation.

The pharmaceutical formulations of the present invention include thosesuitable for oral, parenteral (including subcutaneous, intradermal,intramuscular and intravenous) and rectal administration although themost suitable route will probably depend upon, for example, thecondition and identity of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods will known in the art of pharmacy. All methods includethe step of bringing into association the active ingredient, i.e., thecompound or salt and protecting agent of the present invention, with thecarrier.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with a liquid carrieror, a finely divided solid carrier or both, and then, if necessary,forming the associated mixture into the desired formulation. Thepharmaceutical formulations of the present invention suitable for oraladministration may be presented as discrete units, such as a capsule,cachet, tablet, or lozenge, each containing a predetermined amount ofthe active ingredient, as a powder or granules; as a solution or asuspension in an aqueous liquid or a non-aqueous liquid such as a syrup,elixir or a draught, or as an oil-in water liquid emulsion or awater-in-oil liquid emulsion. The formulation may also be a bolus,electuary or paste.

Generally, a tablet is the most convenient pharmaceutical formulationsuitable for oral administration. A tablet may be made by compressing ormoulding the active ingredient with the pharmaceutically acceptablecarrier. Compressed tablets may be prepared by compressing in a suitablemachine the active ingredient in a free-flowing form, such as a powderor granules, in admixture with, for example, a binding agent, an inertdiluent, a lubricating agent, a disintegrating agent and/or a surfaceactive agent. Moulded tablets may be prepared by moulding in a suitablemachine a mixture of the powdered active ingredient moistened with aninert liquid diluent. The tablets may optionally be coated or scored andmay be formulated so as to provide slow or controlled release of theactive ingredient.

The pharmaceutical formulations of the present invention suitable forparenteral administration include aqueous and non-aqeuous sterileinjection solutions which may contain, for example, an anti-oxidant, abuffer, a bacteriostat and a solution which renders the compositionisotonic with the blood of the recipient, and aqueous and non-aqueoussterile suspensions which may contain, for example, a suspending agentand a thickening agent. The formulations may be presented in unit-doseor multi-dose containers, for example sealed ampoules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water for injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The pharmaceutical formulations of the present invention suitable forrectal administration may be presented as a suppository containing, forexample, cocoa butter and polyethylene glycol.

Compounds and salts of formula (I) have anti-neoplastic activity in thehuman colon SW480 adenocarcinoma cell culture cytotoxicity tests inwhich representative compounds of the present invention have been shownto be active, and in human breast MCF7 adenocarcinoma cell culture. Ithas thus been established that compounds of the present invention areable to inhibit neoplastic growth. Therefore, compounds and salts of thepresent invention are of use in medicine and in particular in thetreatment of neoplastic growth, including solid tumours such as melanomabreast and colon tumours in mammals. Accordingly, the present inventionyet further provides a method for the treatment of susceptible malignanttumours and leukemia in a animal. e.g., a mammal, which comprisesadministering to the animal a therapeutically effective amount of acompound or salt of the present invention in combination with aprotecting agent. In the alternative, there is also provided a compoundor salt of the present invention in combination with protecting agent.In the alternative, there is also provided a compound or salt of thepresent invention in combination with a protecting agent for use inmedicine and in particular for use in the treatment of a neoplasticgrowth, e.g., malignant tumours.

The present invention also provides the use of a compound of formula (I)or a salt therof in combination with a protecting agent for themanufacture of a medicament for the treatment of neoplastic growth.

The animal requiring treatment with a compound or salt of the presentinvention in combination with a protecting agent is usually a mammal,such as a human being.

The following examples illustrate the preparation and pharmacologicalproperties of representative compounds which are useful in the presentinvention and which demonstrate the invention. These examples areillustrations only and should not be construed as limiting or narrowingthe scope of the invention.

EXAMPLE 1(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-1-oxo-2-isoindolinyl)glutaricacid (Compound A)

A. Diethyl (S)-2-(4-nitrophthalimido)glutarate

Diisopropylethylamine (24 ml, 0.138 mole) (Aldrich) was added to asuspension of 4-nitrophthalic anhydride (25 g, 0.13 mole) (Tokyo Kasei)and L-glutamic acid diethyl ester hydrochloride (35 g, 0.146 mole)(Aldrich) in toluene (130 ml). The reaction mixture was stirred atreflux utilizing a Dean-Stark trap for 2.5 hours. After cooling, thesolution was diluted with diethyl ether (300 ml), washed with water (75ml), saturated NaHCO₃ solution (50 ml), dried (MgSO₄), and concentratedin vacuo at 70° C. to give diethyl (S)-2-(4-nitrophthalimido) glutarateas an oil that solidified to a white solid on standing (35.8 g).M.P.=65.5-66.5° C.

B. Diethyl (S)-2-(4-aminophthalimido)glutarate

A suspension of diethyl (S)-2-(4-nitrophthalimido)glutarate (35.6 g,94.1 mmol) and 10% palladium on carbon (0.5 g) (Aldrich) in ethanol (200ml) was shaken under a hydrogen atmosphere (40-50 psi) for 26 hours. Thesolution was filtered through celite and concentrated in vacuo. Theresidue was purified by chromatography on silica gel (250 g) elutingwith diethyl ether:hexane (4:1) to give diethyl(S)-2-(4-aminophthalimido)glutarate as a viscous yellow oil (29.1 g).

C. Diethyl (S)-2-(5-amino-1-oxo-2-isoindolinyl)glutarate

A solution of diethyl (S)-2-(4-aminophthalimido)glutarate (10.5 g 30.2mmol) in ethanol (150 ml) was cooled in an acetonitrile/CO₂ bath.Concentrated HCl (25 ml) was added followed by 30 mesh granular Zn (10.5g, 0.161 mole) (Fisher) when the internal temperature has reached −40°C. The reaction mixure was stirred 1.5 hours at this temperature and afurther 1 hour at −10° C. The excess of Zn was filtered from thesolution, 10% palladium on carbon (1.0 g) was added, and the solutionshaken under hydrogen at (30-50 psi) overnight. The catalyst was removedby filtration through celite and the filtrate concentrated in vacuo. Theresidue was absorbed onto silica gel (15 g) and purified bychromatography on silica gel (440 g) eluting with ethylacetate:methylene chloride (1:14 g).

D 9-Bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one

To a hot solution of 3,9-dimethylbenzo[f]quinazolin-1(2H)-one (4.00 g,17.9 mmol) in benzene (2000 ml) under nitrogen was addedN-bromo-succinimide (4.00 g, 22.5 mmol). The reaction mixture wasstirred just below reflux for 30 minutes, then at a gentle reflux for 30minutes. The resulting suspension was allowed to cool for 2 hours, thesolid filtered and dried at 70° C. under reduced pressure to give9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one (4.32 g, 83% purityby NMR).

E. Diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate

A solution of crude 9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one(4.32 g), (S)-diethyl 2-(5-amino-1-oxo-2-isoindolinyl) glutarate (4.0 g,12 mmol), and NaHCO₃ (2.0 g, 24 mmol) in DMF (30 ml) was stirred undernitrogen at 105° C. for 1.5 hours. After cooling, acetic acid (1 ml, 17mmol) was added, the reaction mixture transferred to a larger roundbottom flask with ethanol, and then concentrated in vacuo onto silicagel (30 g). The absorbed material was purified by chromatography onsilica gel eluting with methanol:methylene chloride (1:24) and thenprecipitation of the solid from methylene chloride (˜20 ml) with ethylacetate (45 ml) and methanol (5 ml). The white solid was filtered undernitrogen and dried under high vacuum to give diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]-quinazolin-9-yl)-methyl)amino)-1-oxo-2-isoindolinyl)glutarate(3.27 g).

F(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

A solution of diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxo-benzo-[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate(3.20 g, 5.75 mmol) in 0.2 N NaOH (140 ml) was stirred under nitrogenfor 3 hours at room temperature. The solution was then slowly adjustedto pH3 with 1 N HCl and the resulting suspension allowed to stirbriefly. The white solid was filtered under nitrogen, washed with waterand dried under high vacuum to give(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid (2.85). ¹H NMR (DMSO-d₆/D₂O, 300 MHz) δ: 1.86-2.34 (m, 4H, glu CH₂,CH₂), 2.44 (s, 3H, CH₃), 4.25 (very strongly coupled AB pair, 2H, gluNCH₂Ar), 4.58 (s, 2H, C⁹—CH₂), 4.67-4.75 (m, 1H, glu CH), 6.69-6.77 (m,2H, Ar), 7.37 (d, J=8 Hz, 1H Ar), 7.59 (d, J=9 Hz, 1H, Ar) 7.64 (dd,J=8.2 Hz 1H, Ar), 8.01 (d, J=8 Hz, 1H, Ar), 8.22 (d, J=9 Hz, 1H, Ar),9.85 (s, 1H, Ar). Anal Calculated for C₂₇H₂₄N₄O₆. 1.6H₂O: C, 61.27; H,5.18; N,10.58. Found: C 61.29; H, 5.18; N, 10.57.

EXAMPLE 2(s)-2-(5-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

A. N-(9-Bromomethyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide

N-(9-methyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide (15 g,48 mmol) was dissolved in refluxing benzene (4000 ml). The reaction wasremoved from heat and N-bromosuccinimide (11.28 g, 64 mmol, Kodak)added. The solution was heated under reflux for 2 hours. Benzene wasremoved in vacuo, the residue slurried with a small volume of ethanol,filtered and dried under high vacuum to give the bromomethyl derivative.The product was used without further purification.

BDiethyl(S)-2-(5-(((1,2-dihydro-1-oxo-3-pivalamidobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate(S)-diethyl 2-(5-amino-1oxo-2-isoindolinyl)glutarate (2.8) andN-(9-Bromomethyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide(2.0 g) were heated in dimethylformamide (15 ml) at 115° C. for 1.5hours. The reaction mixture was concentrated onto silica gel, elutingwith methylene chloride/methanol (97:3). The fractions containingproduct were evaporated and stored under high vacuum overnight. Thepartially crystallized residual oil was suspended in ethyl acetate andfiltered. The solid was recrystallized from methanol, filtered and driedunder high vacuum to yield the diester (0.21 g) as a white solid.

C.(S)-2-(5-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

A solution of diethyl (S)-2-(5-(((1,2-dihydro-1-oxo-3-pivalamidobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate in 0.33M aqueous sodium hydroxide/tetrahydrofuran (12 ml, 1:1) was heated toreflux. Additional water (6 ml) was added to keep the reaction mixturehomogeneous, and the solution was heated under reflux for 2.5 hours. Thecooled solution was adjusted to pH 3 with 1M hydrochloric acid, theprecipitate filtered off, washed with water and dried under high vacuum.The crude product, a white solid (0.15 g), contained approximately 20%of the 3-pivalamide of the desired diacid by NMR. The solid wasredissolved in 0.5 M sodium hydroxide (5 ml) and stirred at roomtemperature for 2 days. The pH of the solution was adjusted to 2, theprecipitated-solid filtered off, washed with water, and dried at roomtemperature under high vacuum to yield the diacid as a white solid (0.13g). ¹H NMR (DMSO-d₆, 300 MHz); δ1.88-2.08 (m, 1H), 2.10-2.30 (m,3H),2.26 (s, 2H), 4.52 (d, J=5.6 Hz, 2H), 4.65-4.75 (m, 1H), 6.57 (br s,2H), 6.67-6.75 (overlapping s and dd, 2H), 7.13 br t, J=5.6 Hz, 1H),7.27 (d, J=8.9 Hz, 1H) 7.35 (d, J=8.3 Hz, 1H), 7.46 (dd, J=8.3, 1.1 Hz,1H), 7.85 (d,j=8.2 Hz, 1H), 8.00 (d, J=8.9 Hz, 1H), 9.68 (s, 1H), 11.14(brs, 1H), 11.9-12.9 (v br s 2H. Anal. Calculated for C₂₈H₂₃N₅O₆.2.5H₂O: C. 57.61 H, 4.93; N, 12.80.

EXAMPLE 3N-4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

A.Diethyl-N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamate

N-(4-aminobenzoyl)-L-glutamic acid diethyl ester (6.06 g, 0.0188 mole)(Aldrich) and1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]-quinazolin-9-sulfonyl chloride(5.84 g, 0.0188 mole) were dissolved in pyridine (55 ml) and thereaction mixture stirred at room temperature for 3.5 hours. The pyridinewas removed in vacuo, the residue washed with water, and the pink solidcollected by filtration. The crude product was dried under high vacuum,then subjected to chromatography on a Waters Prep 500 instrument (silicaCartridge, elution with methanol: methylene chloride (1:4). The productwas recrystallized from ethanol and dried under high vacuum to yield thediethyl ester (5.68 g, 51%).

B.N-(4-((1,2,5,6-Tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

Diethyl-N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido) benzoyl)-L-glutamate (4.53 g, 7.6 mmole)was dissolved in N-NaOH (64 ml) and the solution stirred at roomtemperature for 4 hours. The pH of the solution was adjusted to 3.00with 1N HCl, the solid collected by filtration, washed with water anddried under high vacuum to yield the product as an off-white solid (3.94g 96%). ¹HNMR (DMSO-d₆,300 MHz) δ: 1.84-1.96 (m,1H,glu CH); 2.00-2.10(m, 1H, glu CH); 2.32(s, 3H, CH₃, superimposed over t, 2H, glu CH₂);2.71 (m, 2H, Ar CH₂); 2.89 (m, 2H, Ar CH₂); 4.28-4.36 (m, 1H, glu CH),7.20 (d, J=9 Hz, 2H, Ar); 7.39 (d, J=8 Hz, 1H, Ar); 7.62 (dd, J=8.2 Hz,1H, Ar); 7.74 (d, J=9Hz, 2H, Ar); 8.44 (d, J=8 Hz, 1H, gluNH); 9.21 (d,J=2Hz, 1H, Ar); 10.73 (s, 1H, SO₂NH); 12.36 (br s, 2H, CO₂H); 12.72 (brs, 1H, NH). Anal Calculated for C₂₅H₂₄N₄O₈S. 3/2H₂O: C,52.91; H,4.79;N,9.87; S,5.65. Found: C,52.98; H,4.78; N,9.87; S,5.58.

EXAMPLE 4N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-2-fluorobenzoyl)-L-glutamicacid

A. Diethyl N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-fluorobenzoyl)-L-glutamate

To a hot solution of 3,9-dimethylbenzo[f]quinazolin-1(2H)-one (2.0 g,8.9 mmol) in benzene (1000 ml) under nitrogen was addedN-bromosuccinimide (NBS) (2.0 g, 11 mmol). The solution was stirred atreflux for 1 hour and then concentrated in vacuo to give crude9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one. The solid wassuspended with diethyl N-(4-amino-2-fluorobenzoyl)-L-glutamate (T. R.Jones et al., UK Patent GB 2175 903A, 1986)(6.0 g, 18 mmol) in DMF (20ml) and stirred under nitrogen at 100° C. for 30 minutes. The reactionmixture was allowed to cool. N-methylmorpholine (1.0 ml, 9.1 mmol)(Aldrich) was added, and the solution concentrated under high vacuum.The residue was purified with silica gel chromatography eluting withmethylene chloride:THF (5:1). Fractions containing product wereconcentrated in vacuo to a thick paste, the solid suspended in a smallvolume of diethyl ether, filtered under nitrogen, and dried under highvacuum to give diethylN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamateas a white solid (2.3 g).

B.N-(4-(((2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-2-fluorobenzoyl)-L-glutamicacid

A solution of diethylN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamate(2.3 g, 4.1 mmol) in ethanol (25 ml) and 0.2 N NaOH (100 ml) was stirredunder nitrogen at room temperature for 3 hours. The solution wasadjusted to pH 7 with 1N HCl and reduced in volume under vacuum toremove the ethanol. The product was precipitated by acidifying thesolution with 1N HCl to pH 3 with stirring under nitrogen. Thesuspension was stirred 15 minutes, filtered under nitrogen, washed withwater, pressed with a sheet of latex to remove excess water, and driedunder high vacuum to giveN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quina-zolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid as a white solid (2.1 g. ¹H NMR (DMSO-d₆,300 MHz) d: 1.82-2.12 (m,2H, glu CH₂), 2.29 (t, J=7 Hz, 2H, glu CH₂), 2.43 (s, 3H, C³—CH₃),4.32-4.42 (m, 1H, glu CH), 4.57 (d, J=6 Hz, 2H, C⁹—CH₂), 6.39 (dd,J=15.2 Hz, 1H, Ar), 6.53 (dd, J=9.2 Hz 1H, Ar), 7.30 (t, J=6 Hz, 1H,ArNH), 7.47 (dd, J=9.9 Hz, 1H, Ar), 7.59(d, J=9 Hz, 1H. Ar), 7.61(dd,J=8.2 Hz, 1H, Ar), 7.73 (t, J=7 Hz, 1H, glu NH), 8.01 (d, J=8 Hz, 1H,Ar), 8.22 (d, J=9 Hz, 1H, Ar), 9.85 (s, 1H, Ar), 12.42 (br s, 2H,CO₂H's), 12.53 (s, 1H, N²H) Anal. Calculated for C₂₆H₂₃FN₄O₆.3/2 H₂O.1/3NaCl: C, 56.49: H, 4.74: N, 10.14; Cl, 2.12: Na, 1.37. Found: C, 56.48:H, 4.64; N, 10.20: Cl, 2.01; Na, 1.30.

EXAMPLE 5

The thymidylate synthase inhibitor Compound A (8 mg/kg) was lethal whenadministered to beagle dogs as an iv bolus for five consecutive days. Incontrast Compound A at 20 mg/kg was not lethal when given as an iv bolus30 minutes after oral administration of folic acid at 50 mg/kg.Similarly, five consecutive days of 10 mg/kg of Compound A iv was lethalwhen coven alone. But when the same dose was given 30 minutes after anoral dose of 10, 50, 100 or 200 mg/kg of leucovorin (Wellcovorin) nodeaths occurred.

EXAMPLE 6

Compound A administered as an ip bolus for five days at 3.2 or 10 mg/kgto nude mice bearing the human tumor GC3TK in the subrenal capsuleproduced complete inhibition of tumor growth. When 50 mg/kg of folicacid or 200 mg/kg leucovorin was administered orally 30 minutes prior tothe ip bolus dose of Compound A complete inhibition of tumor growth wasstill observed. In another experiment, in the absence of protectant 10mg/kg Compound A produced complete growth inhibition and 3.2 mg/kgproduced 89% inhibition. When 500 mg/kg folic acid was administeredorally 30 min prior to the Compound A, tumor growth inhibition was notsignificantly different from that observed in the absence of the folicacid.

TABLE Influence of Folic Acid or leucovorin upon toxicity and antitumorefficacy of Compound A [Folic Acid] mg/kg) 0 10 50 100 200 500 10 mg/kgCompound A lethal +/− m m m m Dog tox. bone marrow toxicity Short term:— — Long term: — 10 mg/kg Compound A >100 >100 Mouse antitumor (% I) 3.2mg/kg Compound A  89  78 Mouse antitumor (% I) 10 mg/kg CompoundA >100 >100 Mouse antitumour (% I) 3.2 mg/kg Compound A  100  100 Mouseantiumor (% I) [leucovorin] (mg/kg) 0 5 10 50 100 200 10 mg/kg CompoundA Lethal +/− m m m m Dog tox bone marrow toxicity — — Short term: 10mg/kg Compound A >100 >100 Mouse antitumor (% I) 3.2 mg/kg Compound A 100  100 Mouse antitumor (% I) Key: +/−: one animal died, one hadminimal toxicity m: minimal toxicity —: no toxicity

We claim:
 1. A method of reducing intestinal toxicity associated with the administration of a therapeutically effective amount of a non-competitive thymidylate synthase inhibitor to a mammal, comprising: administering to said mammal a folate derivative selected from folic acid, 5-formyltetrahydrofolate, or 5-methyltetrahydrofolate in an amount effective to reduce said toxicity without blocking therapeutic effect of the non-competitive thymidylate synthase inhibitor, wherein the non-competitive thymidylate synthase inhibitor is (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid.
 2. A method as claimed in claim 1, wherein the folate derivative is administered either, before, concurrentlt, with, or after the administration of the thymidylate synthase inhibitor.
 3. A method of reducing intestinal toxicity associated with the administration of a therapeutically effective amount of a non-competitive thymidylate synthase inhibitor to a mammal, comprising: administering to said mammal folic acid in an amount effective to reduce said toxicity without blocking therapeutic effect of the non-competitive thymidylate synthase inhibitor, wherein the non-competitive thymidylate synthase inhibitor is (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid.
 4. A method of treating a susceptible tumor in a mammal, comprising: administering to said mammal a therapeutically effective amount of (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid and folic acid in an amount effective to reduce said toxicity without blocking therapeutic effect of the (S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaric acid. 